Activatable cell penetrating peptides for imaging protease activity in vivo

Aberrant protease activity is a shared feature of most degenerative pathophysiologies, and could potentially have diagnostic as well as prognostic value several major diseases such as cancer, atherosclerosis and stroke if it could be visualized clinically. Here we have designed and validated a new methodology for looking at protease activity in live animals. In their simplest form, activatable cell penetrating peptides (ACPP's) are hairpin shaped molecules that are comprised of a polycationic cell penetrating peptide, a neutralizing polyanion separated by a protease cleavable linker. In the presence of protease, the linker is cleaved and the released polycation can freely associate with cells. To detect these molecules in vivo, a payload such as a fluorophore or a T1 imaging agent is attached to the polycation. We have found that MMP cleavable ACPP's give fluorescence contrast ratios of up to 3-fold in tumors and atherosclerosis and thrombin cleavable agents give up to ten-fold contrast in stroke. Uptake is inhibited by commercially available MMP and thrombin inhibitors as well as by genetically knocking out MMP's. Attaching a large molecular weight carrier causes accumulation of fluorophores to increase further, and by attaching several gadolinium chelates to a single carrier it is possible to deliver micromolar amounts of gadolinium to tumors, allowing visualization by T1 MRI without loss of protease dependence. A dual labeled probe allows localization of tumor proteases by both MRI and fluorescence, and may have clinical utility for image guided surgery. We conclude that ACPP's represent a valid approach for detecting activity of specific proteases in a variety of degenerative disease processes, and may even be useful in the clini